Enzymes that are L-glutaminases exhibit antitumor activity. Thus, work to develop these enzymes into therapeutic agents is in progress. Present limitations include the enzymes' substrate affinity, size, immunogenicity, and, in some cases, dual function as both L-glutaminase and L-asparaginase. The objective of this project is to obtain a single-function L-glutaminase that is smaller in size. Phase I includes both the cloning and expression of the gene encoding Pseudomonas 7A L-glutaminase-L-asparaginase, an enzyme with very high affinity for L-glutamine. Not only will Phase I work provide a larger and cheaper source of enzyme for clinical trials, but it will also allow the pursuit of the second phase of the project which is to obtain, through oligonucleotide and deletion mutagenesis, and enzyme that is exclusively an L-glutaminase and that is sufficiently small to allow for improved penetrability of tumors located in the extravascular space. The antitumor activity of the resultant genetically-engineered enzymes will be tested by injecting them into tumor-bearing mice. Depending upon the similarities between prokaryotic and eukaryotic L-glutaminases, the cloned Pseudomonas 7A gene might also serve as a probe to isolate a human, nonimmunogenic, L-glutaminase.